1. Field of the Invention
This invention relates to novel strains of non-toxigenic Aspergillus flavus and compositions containing the strains and to methods for the preharvest control of Aspergillus-related toxins in crops.
2. Description of the Related Art
Aflatoxins produced by the fungi Aspergillus flavus and Aspergillus parasiticus are among the most carcinogenic compounds known from nature and are also acutely hepatotoxic in a variety of animals (Williams et al., American Journal of Clinical Nutrition, Volume 80, 1106-1122, 2004). These mycotoxins account for enormous monetary losses worldwide in crops such as, for example, peanuts, corn, tree nuts, spices, and cottonseed due to the costs associated with aflatoxin monitoring and the market rejection of crops (Robens and Cardwell, The Costs of Mycotoxin Management in the United States, in: Aflatoxin and Food Safety, ed. H. K. Abbas, Boca Raton, Fla.: CRC Press, 1-12, 2004). Regions of the world where aflatoxins are not regulated report periodic outbreaks of aflatoxicosis and associated human deaths (Krishnamachari et. al., Lancet, Volume 1, 1061-1063, 1975; Lye et al., American Journal of Tropical Medicine and Hygene, Volume 53, 68-72, 1995, Azziz-Baumgartner et al., Environmental Health Perspectives, Volume 113, 1779-1783, 2005).
Aflatoxin contamination of peanuts, corn, and cottonseed can be effectively reduced through biological control measures in which large amounts of a non-toxigenic A. flavus strain are applied to fields (Dorner, Biological Control of Aflatoxin Crop Contamination, in: Aflatoxin and Food Safety, ed. H. K. Abbas, Boca Raton, Fla.: CRC. Press, 333-352, 2005). High populations of the applied non-toxigenic strain compete with native aflatoxigenic populations during crop invasion and thereby reduce aflatoxin contamination. Inoculum consisting of either conidia-coated or minimally colonized grain (Bock and Cotty, Biocontrol Science and Technology, Volume 9, 529-543, 1999; Dorner, Peanut Science, Volume 36, 60-67, 2009, Domer et al., U.S. Pat. No. 6,306,386, issued Oct. 23, 2001; both herein incorporated by reference in their entirety) is applied during crop maturation to the soil surface where moisture absorption results in extensive sporulation and subsequent conidial dispersal of the non-toxigenic strain into soil and onto the plants. In corn, aerial application of inoculum onto developing ears appears to be more effective than the traditional method of soil application (Dorner, Journal of Food Protection, Volume 72, 801-804, 2009; Lyn et al., Food Additives and Contaminants, Volume 26, 381-387, 2009).
Two major non-toxigenic strains of A. flavus are currently registered through the United States Environmental Protection Agency for use in biological control of aflatoxins: AF36, which is approved for use on cotton (U.S. Environmental Protection Agency, 2003) and NRRL 21882, which is the active ingredient in AFLA-GUARD® for application to peanuts (U.S. Environmental Protection Agency, 2004) and whose use was later approved for corn. Aflatoxins are synthesized by a gene cluster consisting of approximately 25 genes (Yu et al., Applied and Environmental Microbiology, Volume 70, 1253-1262, 2004). Cyclopiazonic acid (CPA), an unrelated mycotoxin produced by A. flavus, is synthesized by a gene cluster adjacent to the aflatoxin gene cluster (Chang et al., Fungal Genetics and Biology, Volume 46, 176-182, 2009). Nonproduction of aflatoxins in AF36 is due to a single nucleotide polymorphism in the polyketide synthase gene early in the aflatoxin pathway (Ehrlich and Cotty, Applied Microbiology and Biotechnology, Volume 65, 473-478, 2004), whereas NRRL 21882 is missing the entire aflatoxin and CPA gene clusters (Chang et. al., Fungal Genetics and Biology, Volume 42, 914-923, 2005; Chang et al., 2009, supra). Other A. flavus strains that do not produce aflatoxins contain various nucleotide polymorphisms and partial deletions in the aflatoxin gene cluster (Chang et al., 2005, supra; Donner et al., Food Additives and Contaminants, Volume 27, 576-590, 2010).
Fungal vegetative compatibility, or the ability of strains to form stable hyphal fusions with each other, is controlled by a series of het loci whose genes must all be identical for stable fusions to occur (Leslie, Annual Review of Phytopathology, Volume 31, 127-150, 1993). Populations are often divided into subpopulations called vegetative compatibility groups (VCGs) consisting, of vegetatively compatible individuals. Because the het loci are scattered throughout the genome, VCGs provide an effective multilocus measure of genetic diversity within populations. In A. flavus populations, strains vary greatly in their capacity to produce aflatoxins and CPA, and most of this variation can be attributed to differences among VCGs (Horn et al., Mycologia, Volume 88, 574-587, 1996). The non-aflatoxin-producing component of A. flavus populations also exhibits high genetic diversity based on the large number of VCGs (Horn and Dorner, Applied and Environmental Microbiology, Volume 65, 1444-1449, 1999).
Cotty (U.S. Pat. No. 5,171,686, issued Dec. 15, 1992 and U.S. Pat. No. 5,294,442, issued Mar. 15, 1994) discloses a non-toxigenic strain of A. flavus which inhibits aflatoxin production by toxigenic strains. The patent teaches that agricultural commodities inoculated simultaneously with both a non-toxigenic strain and a toxigenic strain produce seed with up to 100-fold less aflatoxin than commodities inoculated with a toxigenic strain alone. The patent only discloses that the patented strain fails to produce aflatoxin. There is no disclosure of its lack of ability to produce other toxins such as, for example, CPA.
Cole et al. (U.S. Pat. No. 5,297,661, issued Mar. 8, 1994) and Dorner et al. (Journal of Food Protection, Volume 55, 888-892, 1992) disclose a non-aflatoxigenic strain of A. parasiticus. The references teach the use of this strain as a biocontrol agent which reduces aflatoxin contamination of soil-borne crops.
Phildain et. al. (International Journal of Food Microbiology, Volume-93, 31-40, 2004), Takahashi et al. (Journal of Food Protection, Volume 67, 90-95, 2004), Tran-Dinh et al. (Mycological Research, Volume 103, 1485-1490, 1999), and Wei and Jong (Mycopaihologia, Volume 93, 19-24, 1986) all disclose non-toxigenic strains of A. flavus wherein the strains do not produce aflatoxin.
Horn et al. (1996, supra) and Horn and Dorner (1999; supra) disclose isolates of A. flavus that fail to produce the mycotoxins aflatoxin and CPA.
While various strains of non-toxigenic Aspergillus for control of toxigenic fungi are known in the art, there still remains a need for effective biocontrol agents for toxigenic fungi. The present invention described below includes non-toxigenic strains of Aspergillus, especially non-toxigenic strains of A. flavus, which are antagonistic to toxigenic fungi. The present invention also provides a method for controlling toxigenic fungi in agricultural crops which is different from the related art methods.